Combined treatment with antibody against CD40 ligand and one transfusion of donor splenocytes prolonged survival of fully mismatched BALB/c skin allografts on C57BL/6 recipients, with approximately 20% of grafts surviving > 100 days. In vitro alloresponsiveness in treated animals was reduced in the immediate post-transplantation period, but by day 100 was increased despite the presence of a successful allograft. The presence of alloreactivity on day 100 was confirmed in vivo by adoptive transfer, which suggests that our protocol had induced either a state of "split tolerance" or "graft accommodation." Mice with skin grafts that had survived for > or = 100 days revealed no evidence of lymphoid chimerism. Treatment with donor splenocytes and antibody against CD40 ligand permits long-term survival of highly antigenic donor skin allografts despite the presence of functionally intact alloreactive lymphocytes.